Higher plants are obligately aerobic organisms, that is, they require oxygen for survival and growth. Natural environmental conditions would never expose a plant to total anaerobiosis, but temporary flooding or waterlogging of soil can lead to anoxia in the root zone. Plants have evolved adaptive responses to survive the stress of transient anaerobic conditions. Some plants, such as rice, are tolerant of prolonged flooded conditions due to specialized tissue which transfers oxygen from the upper portions of the plant to the submerged root tissue; this specialized tissue is known as the aerenchyma. Maize is an example of a species which tolerates relatively short-term flooding. A specific set of proteins is induced in plants during anaerobic conditions which affect changes in the energy metabolism of the root cells to fit the altered environmental conditions.
The plant anaerobic response has been well studied over the last several years. M. Sachs et al. (1980), Cell 20:761-767, described the patterns of anaerobic protein synthesis in maize seedling roots; relatively few proteins were synthesized, and similar patterns were observed in rice, sorghum, tragopogon, barley, peas, and carrots. R. Okimoto et al. (1980), Planta 150:89-94, extended these observations and demonstrated that although the patterns of aerobic protein synthesis in the various plant tissues were very different, the profiles of proteins synthesized in those tissues under anaerobic conditions were very similar. In maize there are about ten major and ten minor anaerobic proteins (ANPs). Because the ANPs synthesized in a variety of plants are similar and because synthesis displays little tissue specificity, it appears that these proteins are the products of a set of genes whose expression is induced in response to adverse environmental conditions, and that the ANPs may be analogous to the heat shock proteins induced by thermal stress.
The importance of alcohol dehydrogenase in the survival of maize during temporary anaerobiosis has long been known. D. Schwartz (1969), Amer. Nat. 103:479-481, demonstrated that ADH+and ADH-seeds germinated equally well unless the seeds had been exposed to anaerobic conditions. It was later shown that there were two genes encoding alcohol dehydrogenase in maize, Adh1 and Adh2. Both Adh1 and Adh2 are induced after the onset of anaerobiosis (M. Freeling (1973), Mol. Gen. Genet. 127:215-227). Of the two enzymes, Adh1 is the one of primary importance during anaerobic conditions (M. Freeling and D. Schwartz (1973), Biochem. Genet. 8:27-36). During anaerobiosis, oxygen is no longer available to serve as the terminal electron acceptor. Cells adapt to this by increasing glycolysis and turning to ethanolic fermentation, rather than allowing lactic acid to accumulate in the tissue. J. Roberts et al. (1984), Proc. Nat. Acad. Sci. USA 81:3379-3383, and 6029-6033, have shown that ADH- cells undergo a severe drop in intracellular pH due to leakage of protons from the vacuole; in ADH+ cells there is only a slight drop in the intracellular pH. It is believed that the severe decline in intracellular pH is what causes death in the ADH- cells.
The functions of several of the other ANPs of maize have been described. Sucrose synthase, formerly known as ANP87 (87 kd protein) has recently been identified by Freeling and Bennett (1985) Ann. Rev. Genet. 19:297-323. Sucrose synthase is encoded by the Sh1 gene of maize, which has recently been cloned and sequenced (Werr et al. (1985), EMBO J. 4:1373-1380). In anaerobic roots, sucrose synthase catalyzes the hydrolysis of sucrose to fructose and glucose to supply hexose to the cells for glycolysis and energy generation. Two other ANP enzymes which participate in glycolysis are phosphohexose isomerase (P. Kelley and M. Freeling (1984), J. Biol. Chem. 259:673-677) and fructose 1,6-diphosphate aldolase (K. Wignarajah and H. Greenway, (1976), New Phytol. 77:575-584; P. Kelley and M. Freeling (1984), J. Biol.Chem. 259:14180-14183). A sixth identified ANP is pyruvate decarboxylase (K. Wignarajah and H. Greenway, supra; A. Laszlo and P. St. Lawrence (1983), Mol. Gen. Genet. 192:110-117).
Maize Adhl has been cloned and sequenced (E. Dennis et al. (1984), Nucleic Acids Res. 12:3983-4000) as has been Adh2 (E. Dennis et al. (1985), Nucleic Acids Res. 13:727-743). Pea Adhl has recently been cloned and sequenced as well D. Llewellyn et al. (1987) J. Mol. Biol. 195:115-123). The availability of sequence information for the pea gene and several of the maize genes and the growing body of knowledge that nucleotide sequences in the 5' untranscribed regions of both eukaryotic and prokaryotic genes regulate gene expression have led to a search for the sequence information which directs anaerobic induction. Therefore, sequences upstream of several of these genes have been compared with the goal of finding the anaerobic regulatory element(s).
Sequences governing eukaryotic gene expression are the subject of intensive study. Promoter sequence elements include the TATA box consensus sequence (TATAAT), which is typically positioned 20 to 30 bp upstream of the transcription start site. By convention, the start (or cap) site is called +1, and sequences extending in the 5' (upstream) direction are given negative numbers; thus the TATA box would be in the vicinity of -20 to -30. In most instances the TATA box is required for accurate -80 and -100, there can be a promoter element with transcription initiation. Further upstream, often between homology to the consensus sequence CCAAT (R. Breathnach and P. Chambon (1981), Ann. Rev. Biochem. 50:349-383). In plants there can be instead an AGGA element, in which there is a symmetry of adenines surrounding the trinucleotide G(orT)NG (J. Messing et al. (1983), in Genetic Engineering in Plants, eds. T. Kosuge, C. Meredith, and A. Hollaender, pp 211-227).
In animal and yeast systems there is a relatively large body of knowledge describing sequences not necessarily within the promoter which modulate gene expression. One such sequence element is the enhancer, which is defined by G. Khoury and P. Gruss (1983), Cell 33:313-314, as a sequence which increases transcriptional efficiency in a manner relatively independent of position and orientation with respect to a nearby gene. The prototype enhancer is the 72 bp tandem repeat of SV40, which contains the core consensus sequence GGTGTGGAAA(or TTT)G. Generally enhancers in animal systems can act from a position either 5' or 3' to the gene, and can act over distances of one or more kb. In yeast there are sequences located 5' to the transcriptional start site known as upstream activating sequences (UAS's), which may also carry regulatory information. Like the animal enhancers, the yeast UAS's can function in either orientation; however they do not appear to stimulate transcription when placed 3' to the transcription start site (B. Errede et al. (1985), Proc. Nat. Acad. Sci. USA 82:5423-5427; L. Guarente and E. Hoar (1984), ibid. 81:7860-7864; G. Roeder et al. (1985), ibid. 82:5428-5432; K. Struhl (1984) ibid. 7865-7869).
Recently some data have appeared regarding enhancer-like sequences in plant and plant virus genes. J. sequences between -105 and -46 are required for maximal expression of the Cauliflower Mosaic Virus (CaMV) 35S promoter. Contained within that region is a sequence partially homologous to the animal enhancer core consensus sequence, but its functionality has not yet been established. There are other examples of sequences in plant DNA with homology to the animal core consensus sequence, including one in the 5' untranscribed region of the pea legumin gene (G. Lycett et al. (1984), Nucleic Acids Res. 12:4493-4506) and another upstream of the Antirrhimun majus chalcone synthase gene (H. Kaulen et al. (1986), EMBO J. 5:1-8), and several similar sequence motifs located 5' to light-regulated ribulose bis-phosphate carboxylase small subunit genes in Petunia, pea, and tobacco (reviewed by R. Fluhr et al. (1986), Science 232:1106-1112). In none of these cases has it been demonstrated that these plant sequences with homology to the animal enhancer core consensus sequence have activity.
Definitive experiments regarding a plant-active transcription activating element have been performed by J. Ellis et al. (U.S. patent application Ser. No. 011,614, filed Feb. 6, 1987 now pending). A sequence partially homologous to the SV40 core consensus sequence was found in the 5' region of the Agrobacterium tumefaciens T-DNA gene encoding octopine synthase (ocs), which is expressed in infected plant cells and tissues. Deletion of this SV40-like sequence was however shown to have no effect on the level of expression of a downstream gene. It was established, however, that a 16 bp palindromic sequence (5'-ACGTAAGCGCTTACGT-3') not part of the SV40 homologous region was necessary as a part of the ocs-derived fragment or sufficient when inserted as a chemically synthesized oligonucleotide to give transcriptional activation.
There are other sequences known to be present in the 5' flanking regions of genes which regulate the expression of those genes in response to environmental signals. Thermal stress elicits the expression of a family of genes called the heat shock proteins (HSPs); gene induction by elevated temperature requires the presence of a characteristic sequence motif, or heat shock element (HSE). The consensus sequence for the HSE is 5'-CTGAAT-TTCTAGA-3' (H. Pelham and M. Bienz (1982), in Heat Shock: From Bacteria to Man, eds. M. Schlessinger, M. Ashburner, and A. Tissieres, Cold Spring Harbor Laboratory, pp. 43-48). These sequences interact with a heat shock specific transcription factor which allows the induction of the HSP genes (C. Parker and J. Topol (1984), Cell 37:273-283). It is likely that a similar mechanism functions in higher plants because sequences with significant homology to the above HSE have been located in the 5' flanking regions of several soybean heat shock genes (F. Schoffl et al. (1984), EMBO J. 3:2491-2497; E. Czarnecka et al. (1985), Proc. Nat. Acad, Sci. USA (1985) 82:3726-3730; Key et al., U.S. patent application Ser. No. 599,993, filed Apr. 13, 1984). In maize the hsp70 gene has been cloned and sequenced, and carries two copies of the HSE 5' to the transcription start site (D. Rochester et al. (1986), EMBO J. 5:451-458).
Metallothioneins are another class of proteins whose synthesis is induced by stress: in this case by exposure to (toxic) heavy metals in the environment. These genes have in their 5' untranscribed regions copies of a DNA sequence motif called the metal regulatory element (MRE) (reviewed by D. Hamer (1986), Ann. Rev. Biochem. 55:913-951). The mammalian consensus sequence is 5'-TGCGCYCGGCCC-3'. These genes have been well studied in mammalian and yeast systems, but there is no sequence data for cabbage, tomato, and tobacco.
In plants there is a growing interest in the cis-acting sequences which mediate light-regulation and tissue specificity. M. Timko et al. (1985), Nature 318:579-582, described experiments in which the -973 to -90 region of 5' flanking DNA was active, in either orientation, in stimulating expression from the pea ribulose bis-phosphate carboxylase small subunit rbcS ss3.6 gene promoter after illumination. J. Simpson et al. (1985), EMBO J. 4:2723-2729, presented evidence that the 400 bp preceding the pea chlorophyll a/b binding AB80 protein gene carried the sequence information necessary for light regulation and tissue specificity. R. Fluhr et al. (1986), Science 232:1106-1112, showed that the -327 to -46 region 5' to the rbcS-3A gene of pea conferred tissue specificity and light response on a heterologous promoter. The -317 to -82 region of the rbcS-E9 gene gave similar results. The exact sequences directing these responses have not yet been defined.
The existence of known sets of anaerobically induced proteins in several different plant species has led to the hypothesis that there may be a cis-active sequence or sequences which occurs 5' to the structural genes within the ANP group. The availability of sequence information for the maize Adhl (E. Dennis et al. (1984), Nucleic Acids Res. 12:3983-4000), maize Adh2 (E. Dennis et al. (1985), Nucleic Acids Res. 13:727-743), maize Sh1 (W. Werr et al. (1985), EMBO J. 4:1373-1380), pea Adh1 (D. Llewellyn et al. (1986), manuscript submitted), and Arabidopsis Adh (C. Chang and E. Meyerowitz (1986), Proc. Nat. Acad. Sci USA 33:1408-1412), has prompted a comparison of 5' flanking regions with the goal of identifying conserved sequences which might play a regulatory role in the anaerobic induction of those genes.
In D. Llewellyn et al. (1985), in Molecular Form and Function of the Plant Genome, eds. L. Vloten-Doten, G. Groot, and T. Hall, New York: Plenum Press, pp. 593-607, and in E. Dennis et al. (1985), Nucleic Acids Res. 13:727-743, one sequence proposed to be involved in anaerobic regulation of maize Adh1 and Adh2 is 5'-CACCTCC-3'. Interestingly, this sequence is 80% homologous to the complement of the animal enhancer core consensus sequence. M. Freeling and D. Bennett (1985), Ann. Rev. Genet. 19:297-323, speculated that the sequence 5'-TGGGG-3', present in multiple copies upstream of the two maize Adh genes, might be a potential regulatory sequence. They also noted that this sequence was found in association with a sequence similar to the animal enhancer core consensus sequence.
In Llewellyn et al. (1985), supra, there were also two other sequences, with partial homologies 5' to the maize Adh1 and Adh2 genes. In that publication they stated that there was no experimental evidence that these sequences functioned to regulate gene expression. In the present work provides the first documentation that portions of these sequences function in the anaerobic response. The present work identifies sequences in maize Adh1, Adh2 and aldolase gene 5' untranslated regions that confer anaerobic regulation on heterologous genes placed under their control. These anaerobic regulatory sequences function for anaerobic gene expression in plant species other than maize. The DNA sequences, DNA fragments containing them and constructions of the present invention will enable others to selectively express structural genes under anaerobic conditions in plant tissue.